Chronic and infected wounds significantly affect the quality of life for millions of US citizens and are associated with increased risk of complications including the devastating consequence of amputation. As of 2006, infections of diabetic foot ulcers resulted in 82,000 amputations each year and it has been estimated that these numbers will increase by 10% annually. Moreover, in an extensive study of several types of chronic wounds including diabetic foot ulcers, venous leg ulcers, and pressure ulcers, 60% were found to contain bacterial biofilms. The National Institutes of Health estimates that 80% of human infectious diseases are directly related to bacterial biofilm formation. Furthermore, it is widely recognized that bacterial biofilms are highly resistant to antibiotic treatment. There is a growing body of evidence indicating that biofilm formation acts as a major impediment to the healing of cutaneous wounds. Accordingly, there is an urgent need for novel strategies to disrupt biofilm formation in these wounds. The specific aims for this Fast-Track SBIR translational research proposal are designed to advance the development of ExpressGraftEnhance tissue, an antimicrobial living skin substitute created by Stratatech Corporation that has been shown to inhibit the growth of multidrug-resistant bacteria in an in vivo burn infection model (Mol Ther. 2009 Mar;17(3):562-9). Stratatech has established an interdisciplinary series of collaborative partnerships with industry, academia, and the U.S. armed forces to test ExpressGraftEnhance its effectiveness in preventing or disrupting biofilm formation by multidrug-resistant bacteria. Phase I milestones will 1) demonstrate that hCAP-18/LL-37 secreted by ExpressGraftEnhance is efficacious against biofilm formation in vitro, 2) develop potency assays to monitor this bioactivity and 3) optimize ideal production and storage conditions for this living skin substitute tissue. The Phase II study will: 1) determine the efficacy ExpressGraftEnhance tissue in preventing or disrupting bacterial biofilm formation in vitro, 2) demonstrate the efficacy of ExpressGraftEnhance tissue against biofilm formation in both murine and porcine in vivo wound models of biofilm infection, 3) implement and qualify cGMP manufacturing processes and potency assays for ultimate clinical use and 4) demonstrate that late passage cells used to create ExpressGraftEnhance are karyotypically stable, non-tumorigenic, free of viral adventitious agents, and maintain transgene stability and integrity. Stratatech's ultimate goal is to commercialize the ExpressGraftEnhance skin substitute for use in treating cutaneous wounds that are refractory to healing due to bacterial colonization and biofilm formation by wound pathogens. The studies of this proposal are designed to generate preclinical data that are required to support translation of the ExpressGraftEnhance technology into human clinical trials. Bacterial biofilms have become recently become recognized by the medical community as significant barriers to wound healing, and the National Institutes of Health estimates that 80% of human infectious diseases are directly related to bacterial biofilm formation yet no biological treatment options that specifically target biofilm infection have been developed and marketed. The goal of this translational Fast-Track SBIR proposal is to develop a series of collaborative partnerships to expedite the development of an innovative antimicrobial therapeutic aimed at accelerating the healing of wounds by preventing or disrupting wound biofilms formed by multidrug-resistant bacteria. The development of a cultured human skin substitute specifically engineered to disrupt biofilms would target the needs of the millions of patients afflicted with hard-to-heal skin wounds by both eliminating the impediment to healing while also promoting healing, reducing pain, and reducing the likelihood of other major complications.